‘Omics Analysis and Cellular Engineering for the Overexpression of Membrane Proteins in Mammalian Cells
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Date
2015-12-21
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Johns Hopkins University
Abstract
Membrane proteins (MPs) are a medically relevant type of protein that are involved in a number of diseases and disorders, such as Alzheimer’s, anxiety, and depression. In order to accurately design biotherapeutics that treat these diseases, structural information regarding the protein must be known. Many MPs, however, are large, complexly-folded proteins that span the cell membrane several times. As a result, expression of a sufficient amount of MP to enable structural studies is the main bottleneck in MP structure determination. Thus the goal of the presented thesis was to engineer mammalian cells, particularly HEK293, for enhanced MP production.
The serotonin transporter (SERT) was chosen as a model MP for expression studies, as it has been shown to be a difficult to express MP that is toxic to cells when overexpression is attempted. The most successful method employed to increase the expression of SERT in HEK293 cells was overexpression of an anti-apoptosis gene, Bcl-xL. Overexpression of Bcl-xL led to a statistically significant 1.3-fold increase in the expression of functional SERT, as determined by radioligand binding assay. Analysis of endoplasmic reticulum stress response gene expression indicated that Bcl-xL overexpression alleviates the ER stress response that is triggered by overexpression of SERT.
In addition to membrane protein overexpression studies, our work also focused on characterizing the proteome of HEK293 cells, and comparing HEK293 and Chinese hamster ovary (CHO) cells at the transcriptome level. Gene Ontology annotation and KEGG pathway mapping were performed, and enriched pathways and molecular functions in HEK cells were identified. In addition, insights that could be used for targeted cell and genetic engineering to improve recombinant protein production in HEK and CHO cells were made by directly comparing HEK to CHO at the transcriptome level.
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membrane protein overexpression, mammalian cells, HEK293, CHO, proteomics, transcriptomics, SERT